cells Article Inhibiting the P2X4 Receptor Suppresses Prostate Cancer Growth In Vitro and In Vivo, Suggesting a Potential Clinical Target 1, 1, 1, 1 Jiepei He y, Yuhan Zhou y , Hector M. Arredondo Carrera y, Alexandria Sprules , Ramona Neagu 1 , Sayyed Amin Zarkesh 1, Colby Eaton 1, Jian Luo 2, Alison Gartland 1 and Ning Wang 1,* 1 The Mellanby Centre for Bone Research, Department of Oncology and Metabolism, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK; jhe44@sheffield.ac.uk (J.H.); [email protected] (Y.Z.); hmarredondocarrera1@sheffield.ac.uk (H.M.A.C.); arfsprules1@sheffield.ac.uk (A.S.); rneagu1@sheffield.ac.uk (R.N.); [email protected] (S.A.Z.); c.l.eaton@sheffield.ac.uk (C.E.); a.gartland@sheffield.ac.uk (A.G.) 2 Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China; [email protected] * Correspondence: n.wang@sheffield.ac.uk; Tel.: +44-(0)-114-2159216 These authors contributed equally. y Received: 12 October 2020; Accepted: 18 November 2020; Published: 20 November 2020 Abstract: Prostate cancer (PCa) is the most frequently diagnosed cancer in men, causing considerable morbidity and mortality. The P2X4 receptor (P2X4R) is the most ubiquitously expressed P2X receptor in mammals and is positively associated with tumorigenesis in many cancer types. However, its involvement in PCa progression is less understood. We hypothesized that P2X4R activity enhanced tumour formation by PCa cells. We showed that P2X4R was the most highly expressed, functional P2 receptor in these cells using quantitative reverse transcription PCR (RT-PCR) and a calcium influx assay. The effect of inhibiting P2X4R on PCa (PC3 and C4-2B4 cells) viability, proliferation, migration, invasion, and apoptosis were examined using the selective P2XR4 antagonists 5-BDBD and PSB-12062. The results demonstrated that inhibiting P2X4R impaired the growth and mobility of PCa cells but not apoptosis. In BALB/c immunocompromised nude mice inoculated with human PC3 cells subcutaneously, 5-BDBD showed anti-tumourigenic effects. Finally, a retrospective analysis of P2RX4 expression in clinical datasets (GDS1439, GDS1746, and GDS3289) suggested that P2X4R was positively associated with PCa malignancy. These studies suggest that P2X4R has a role in enhancing PCa tumour formation and is a clinically targetable candidate for which inhibitors are already available and have the potential to suppress disease progression. Keywords: purinergic signalling; P2X4 receptor; prostate cancer; ATP; antagonist; xenograft model; clinical datasets 1. Introduction Prostate cancer (PCa) is the second most frequently diagnosed cancer (more than 1.3 million new cases worldwide in 2018) and the fifth leading cause of cancer deaths (over 350,000 deaths annually) in men [1]. The life-time risk of developing PCa is >10% [2], while the incidence of PCa between the ages of 65 to 74 is as high as 35.3% [3]. The mortality rate also rises with age, and 55% of PCa deaths occur over the age of 65 [1]. With advances in treatments, patients with localized PCa have a very good prognosis (five-year survival rate >99%). However, metastatic PCa patients only have a five-year survival rate just above 30% [2]. More importantly, a trend towards an increased PCa incidence and a doubling of Cells 2020, 9, 2511; doi:10.3390/cells9112511 www.mdpi.com/journal/cells Cells 2020, 9, 2511 2 of 15 mortality is estimated worldwide up to the year 2040, particularly affecting developing countries in Africa, Latin America, the Caribbean, and Asia [1]. Effective treatment strategies, in particular for the suppression of disease progression for the majority of patients that present with clinically localised disease, as well as better diagnostic and prognostic biomarkers, are urgently needed. P2X receptors are a group of ATP-gated nonselective ionotropic channels, currently with seven known mammalian members (P2X1–P2X7) [4,5]. The P2X4 receptor (P2X4R) was first cloned in the late 1990s [6,7] and is a typical P2X receptor, containing two transmembrane domains (TM1 and TM2), an intracellular domain with an N-terminus and C-terminus, and a large extracellular loop domain with three ATP binding sites [8]. P2X4R is one of the most sensitive and ubiquitously expressed P2X receptors [9,10]. P2X4R is prominently expressed in central and peripheral neurons, immune cells, and endothelial cells [11]. At the intracellular level, P2X4R localises on the plasma membrane, as well as in intracellular compartments such as lysosomes and vesicles [12]. This wide distribution indicates important regulatory roles of P2X4R in multiple biological and pathological processes, such as pain [13,14], central nervous system (CNS) disorders [11,15], inflammation [16], and cardiovascular diseases [17,18]. P2X4R are also expressed in many cancer types, including lung, colorectal, bladder cancer, leukaemia, brain tumour, and, most importantly to this study, PCa [19]. Functionally, most studies suggest that P2X4R positively associates with tumourigenesis [20–22]. However, whether and how P2X4R is involved in PCa progression remains to be elucidated. Limited 2+ evidence showed that the antidepressant paroxetine, a P2X4R inhibitor, induced [Ca ]i rises in human PCa cells [23], indicating a functional role of P2X4R in PCa progression [24]. More recently, P2X4R was shown to be involved in TGFβ-1 induced invasiveness and epithelial-to-mesenchymal transition (EMT) in PCa cells [25]. These studies suggested a complex link between P2X4R and PCa activities. Here, we comprehensively define the roles of P2X4R in PCa progression and test the hypothesis that P2X4R activity enhances tumour formation by PCa cells. To achieve this objective, we have first examined the transcriptional expression of P2 receptors in several PCa cells lines and determined whether the expressed P2X4R are functional. PCa cell viability, proliferation, migration, invasion, and apoptosis were examined in PCa cells treated with the P2X4R selective antagonists 5-BDBD and PSB-12062. A xenograft murine model was further used to determine whether the blockade of P2X4R using 5-BDBD impaired the PCa tumour growth in vivo. Finally, using publicly available clinical datasets, the association between P2X4R expression and PCa disease progression was established. 2. Materials and Methods 2.1. Cell Culture The PC3 prostate cancer cell line (Prostate Specific Antigen (PSA) non-expressing, androgen- independent) (ATCC, Manassas, VA, USA) was stably transfected with a firefly luciferase gene luc2 (pGL4.51 [luc2/CMV/Neo] vector, Promega, Madison, WI, USA) using a Gene PulserTM electroporator (Bio-Rad, CA, USA). LNCaP (PSA expressing, androgen-sensitive) were purchased from ATCC, and the LNCaP-derived C4-2B4 strain was supplied by the University of Bern (Bern, Switzerland). All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with antibiotics and foetal bovine serum (FBS) (Sigma Aldrich, St. Louis, MO, USA). All cell lines were genetically profiled by Stem Elite ID system (Promega, Madison, WI, USA), which confirmed their identity (18/18 STRs) and regularly screened for mycoplasma contamination. 2.2. Quantitative Real-Time PCR Total RNA were extracted from PCa cell lines using the ReliaPrep™ RNA Cell Miniprep System (Promega). cDNA was synthesized using SuperScript™ III reverse transcriptase (Invitrogen, Life Technologies) with a 1:1 mix of Oligo(dT) 15 and Random primers (Promega). TaqMan Gene Expression Assays were then used to quantify the expression of P2 receptors with an Applied Cells 2020, 9, 2511 3 of 15 Biosystems 7900HT Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific) (50 ◦C, 2 min, 94.5 ◦C, 10 min, followed by 40 cycles of denaturation at 97.0 ◦C for 30 s and an extension at 59.7 ◦C for 1 min). A complete list of TaqMan probes are shown in Supplementary Table S1. In order to systematically compare all target genes and limit interpolated errors, the same threshold values of 0.2 were used for each gene. The data were then analysed using the cycling threshold (CT) relative quantification method, and genes with CT values < 35 were regarded as transcriptionally expressed [26]. A heatmap showing the gene expression pattern variance was built from reciprocals of CT values, using the ‘GenePattern’ web software (http://www.broadinstitute.org). Each colour patch in the heatmap represents the relative gene expression level, with a continuum of expression levels from dark blue (lowest) to bright red (highest). 2.3. P2X4R Agonist and Antagonists P2X4R agonist adenosine 50-triphosphate (ATP) disodium salt hydrate, competitive antagonist 5-(3-bromophenyl)-1,3-dihydro-2Hbenzofuro [3,2-e]-1,4-diazepin-2-one (5-BDBD), and allosteric antagonist 10-[(4-Methylphenyl)sulfonyl]-10H-phenoxazine (PSB-12062) were all purchased from Sigmal-Aldrich. 50 µM ATP was used in the calcium influx assay in order to maximally activate P2X4R but not P2X7R [27,28]. Both 5-BDBD and PSB-12062 powder were reconstituted in DMSO and then serial diluted with PBS into a stock concentration of 200 µM and 300 µM, respectively. The final working concentrations of 5-BDBD and PSB-12062 were 1.0 µM and 1.5 µM for all in vitro experiments, determined based on previously published IC50 values [28,29]. 2.4. Calcium Influx Assay Fluo-4 Direct™ (Thermo Fisher Scientific) was used to examine the calcium influx in PC3 cells treated with P2X4R specific antagonists (1.0 µM 5-BDBD or 1.5 µM PSB-12062) and agonist (50 µM ATP). Briefly, PC3 cells were seeded at 1 104 cells per well in a 96-well plate and incubated for 24 h × before being treated with P2X4R specific antagonists or vehicle in phenol-free DMEM containing 1 × Fluoro-4-direct reagent for 45 min. The fluorescence (Excitation: 494 nm; Emission: 516 nm) was then read every 1.5 s over a 700 s period at 37 ◦C using the FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, CA, USA), while 50 µM ATP and 1 µM ionomycin (Sigmal-Aldrich) were automatically added at 30 s and 450 s, respectively.